Mucoid strains of Pseudomonas aeruginosa cause chronic infections in the lungs of cystic fibrosis patients and are the major cause of morbidity and death in these patients. The production of large amounts of the exoplysaccharide, often call alginate, is responsible for the mucoid colony morphology of these strains. The molecular mechanism responsible for the conversion of nonmucoid strains of P. aeruginosa to those with a mucoid phenotype is unknown. One goal of this proposal is to identify the molecular genetic basis for the activation of alginate production. This will be accomplished by determining the DNA sequence differences between cloned alginate conversion genes, known as algS(On) and algS(Off), from mucoid and nonmucoid cells. Comparison of algS from clinical and environmental isolates utilizing the polymerase chain reaction will indicate whether the genetic mechanism of alginate conversion is conserved. The DNA sequence, transcriptional start site, and the inferred amino acid homology of the adjacent alginate gene, algT, will be determined. Another goal of this proposal is to ascertain the role of environmental factors in the conversion of nonmucoid strains to the mucoid phenotype. A promoterless antiotic resistance "reporter" gene will be cloned into the alginate algT gene to make an algT transcriptional fusion. Induction and regulation of alginate conversion will be selected for in strains containing the algT fusion by resistance to antibiotics. These algT fusions will be examined in various genetic backgrounds and under various growth conditions determine those factors influencing alginate conversion. This proposal will also assess the role of alginate in the pathogenesis of P. aeruginosa infections. By introduction of the cloned alginate conversion genes into P. aeruginosa infections. By introduction of the cloned alginate conversion genes into P. aeruginosa strains with varying lipopolysaccharide structure, mucoid derivatives of these strains, which differ only in the production of alginate, will be constructed. These isogenic strains will be compared to determine the relative contribution of alginate and lipopolysaccharide to immunity, in both in vitro opsonic-killing assays in vivo protection assays in animal models. These comparisons will shed light on the immunity specific for lipopolysaccharide and alginate. The combination of a molecular genetic approach to study the production of alginate and an immunological characterization of this exopolysaccharide should provide important insights into the regulation of this important virulence factor in P. aeruginosa.